2890 J. Agric. Food Chem., Vol. 44, No. 9, 1996
Miyazawa et al.
refrigerator until the time of administration. The last instar
larvae (average weight, 0.5 g) were moved into new cases (100
larvae/case), and the diet was fed to the larvae in limited
amounts. Groups of 500 larvae were fed for 7 days with 7.0 g
of the diet containing 1, then the artificial diet not containing
1 was fed to the larvae for an additional 3 days. The frass
were collected daily (total 10 days) and stored in a solution of
CH2Cl2 (500 mL).
An a lysis of Meta bolites in Cu ltu r e Med iu m . The
extracts of medium were analyzed by GC. Metabolite 2 was
identified by comparison of its retention time with that of the
identified metabolite, and metabolite 4 was identified by
comparison of its retention time with that of the authentic
sample. No bacteria grew on the control (no frass) medium.
The percentage of metabolites was calculated from the peak
areas in the GC spectra of the metabolites.
Isolation an d Iden tification of Metabolites fr om Fr ass.
The frass were extracted three times with CH2Cl2 (500 mL
each time). The extract solution was evaporated under
reduced pressure, and the 6.1 g of the extract was obtained.
The extract was distributed between 5% NaHCO3 aq. and CH2-
Cl2, the CH2Cl2 phase was evaporated, and the neutral fraction
(3.1 g) was obtained. The neutral fraction was analyzed by
GC-MS, and metabolites 2 and 5 were identified by a com-
parison of established MS data. The alkali phase was acidified
with 1 N HCl and distributed between water and CH2Cl2. The
CH2Cl2 phase was evaporated, and the acidic fraction (2.5 g)
was obtained. The acidic fraction was dissolved in CH2Cl2 (20
mL), and CH2N2 (5 mL) was added to the solution. The
solution was evaporated, and the methylated fraction (2.8 g)
was obtained. The methylated fraction was analyzed by GC-
MS, and metabolites methylated 3 and methylated 6 were
identified by a comparison of established MS data. The
methylated fraction was subjected to silica-gel open-column
chromatography (silica gel 60, 230-400 mesh, Merck) with a
9:1 n-hexane:CHCl3 solvent system, and methylated 3 (1263
mg) was isolated. Methylated 3 was dissolved in 5% NaOH
aq. (10 mL), and the solution was refluxed for 1 h at 100 °C.
The solution was acidified with 1 N HCl and distributed
between Et2O and water. The Et2O phase was evaporated
yielding (1176 mg) of 3. Metabolites 3 and methylated 3 were
identified by a comparison of established MS, IR, and NMR
data.
In jection of r-Ter p in en e (1). The last instar larvae
(average weight, 0.5 g) were placed on ice (0 °C) to be put in
a state of apparent death. Then, 1 mg (1.2 µL) of 1 (no solvent)
was injected into the hemolymph with a microsyringe. The
larvae were warmed back to 25 °C, and rearing was continued.
The abdomen of larvae was cut with a scalpel, with 0.1 mL of
hemolymph in the living body was collected. The concentra-
tion of metabolites in the hemolymph were determined 1, 3,
6, 12, and 24 h after injection of 1. The hemolymph was
acidified with 1 N HCl and distributed between Et2O and a
saturated solution of salt. The Et2O phase was evaporated,
and the extract was obtained.
4-Isop r op yl-1,3-cycloh exa d ien em eth a n ol (2). Electron-
impact MS (EIMS) m/z (rel. int.): 137 [M-Me]+ (5), 127 (40),
119 (42), 109 (100), 81 (45), 43 (88).
4-Isop r op yl-1,3-cycloh exa d ien oic Acid (3). EIMS m/z
(rel. int.): 166 [M]+ (33), 151 (14), 121 (60), 105 (56), 91 (34),
79 (100), 51 (42), 43 (61); IR (νmax cm-1): 3437, 2962, 1667,
1
1634, 1574, 1427, 1283, 1229; H NMR (CDCl3): δ 1.08 (6H,
d, J ) 7.3 Hz, Me-9 and Me-10), 2.22 (2H, t, J ) 12.4 Hz, H-5),
2.40 (1H, m, J ) 7.3 Hz, H-8), 2.45 (2H, m, J ) 12.4 Hz, H-6),
5.86 (1H, m, J ) 5.4 Hz, H-3), 7.15 (1H, m, J ) 5.4 Hz, H-2);
13C NMR (CDCl3): δ 20.8 (t, C-9 and C-10), 21.4 (q, C-5), 25.5
(q, C-6), 35.2 (d, C-8), 116.5 (d, C-3), 124.0 (d, C-2), 136.9 (s,
C-4), 155.9 (s, C-1), 172.8 (s, C-7).
4-Isop r op yl-1,3-cycloh exa d ien oic Acid (3) a s Meth yl
Ester . EIMS m/z (rel. int.): 180 [M]+ (33), 137 (60), 121 (71),
105 (100), 91 (70), 77 (78), 59 (67), 51 (36), 43 (98); IR (νmax
cm-1): 2963, 1708, 1583, 1436, 1268, 1226, 1091; 1H NMR
(CDCl3): δ 1.07 (6H, d, J ) 6.5 Hz, Me-9 and Me-10), 2.20
(2H, t, J ) 10.0 Hz, H-5), 2.36 (1H, m, J ) 6.5 Hz, H-8), 2.45
(2H, m, J ) 10.0 Hz, H-6), 3.74 (3H, s, OMe), 5.82 (1H, m, J
) 5.9 Hz, H-3), 7.01 (1H, m, J ) 5.9 Hz, H-2); 13C NMR
(CDCl3): δ 20.9 (t, C-9 and C-10), 21.9 (q, C-5), 25.5 (q, C-6),
34.2 (d, C-8), 51.9 (t, OMe), 116.4 (d, C-3), 124.7 (d, C-2), 134.6
(s, C-4), 154.4 (s, C-1), 168.0 (s, C-7).
Cu m ic Alcoh ol (5). EIMS m/z (rel. int.): 135 [M-Me]+ (26),
43 (100).
Cu m ic Acid (6) a s Meth yl Ester . EIMS m/z (rel. int.):
178 [M]+ (34), 163 (100), 119 (69), 91 (63), 77 (41), 59 (44), 51
(39).
RESULTS AND DISCUSSION
Metabolites fr om Fr ass. R-Terpinene (1) was mixed
in the diet of larvae at a high concentration to increase
the production of potential metabolites. A concentration
of 1 of 10 mg/g of diet was chosen as an upper limit for
administration and apparently was not toxic to the
larvae. Metabolites in frass were not only end metabo-
lites but also intermediary metabolites at this concen-
tration. The four metabolites isolated or detected from
frass were identified as 4-isopropyl-1,3-cyclohexa-
dienemethanol (2), 4-isopropyl-1,3-cyclohexadienoic acid
(3), cumic alcohol (5), and cumic acid (6). The majority
of metabolites were end metabolites 3 (71.7% in me-
tabolites of 1) and 6 (7.8%), and the remainder were
intermediary metabolites 2 (3.8%) and 5 (0.5%). These
metabolites were produced by oxidation at the 7-position
of 1, and allylic oxidation was the main metabolic
pathway.
Tim e Cou r se. The time course of the in vivo
metabolism of 1 by the larvae was examined to better
understand the metabolic pathways. A time course was
determined by monitoring the concentration of metabo-
lites in the hemolymph over time following the injection
of a large dose of 1 into the larvae (the very large dose,
1 mg/larva, chosen did not affect the life of the larvae).
Minor metabolites could not be detected by GC because
various components contained in the extract from the
hemolymph lowered the metabolite content in the
extract. The hemolymph concentration of 1 and me-
tabolites in the living body are shown in Figure 1.
Starting material 1 disappeared completely from the
hemolymph at 1 h after injection of 1. Following the
disappearance of 1, intermediary metabolite 2 reached
An a lysis of Meta bolites in Livin g Bod y. The extracts
of hemolymph were analyzed by GC, and metabolites 2 and 3
were identified by a comparison of retention times of identified
metabolites. The concentration of metabolites was calculated
from the peak area of GC spectra of the extracts of hemolymph,
with 1 as an internal standard.
In jection of p-Cym en e (4). Same procedure as described
for 1.
In cu ba t ion of In t est in a l Ba ct er ia w it h r-Ter p in en e
(1). Petri dishes, pipets, and solutions were autoclaved.
A
GAM Broth (Nissui Pharmaceutical Company, Ltd., Tokyo,
J apan) was adjusted to pH 9.0 and placed in Petri dishes at
10 mL/Petri dish. The fresh frass (5 g) of the last instar larvae
were suspended in physiological saline (100 mL), and the
suspension (1 mL) was pipetted into the medium. The medium
without frass was also prepared for a blank experiment. These
media were incubated (18 °C, darkness, 2 days) under aerobic
and anaerobic conditions. After some growth of bacteria, 1
(10 mg/Petri dish) was added to the medium, and the incuba-
tion was continued. The percentage of metabolites in the
medium were determined 12, 24, and 48 h after addition of 1.
The medium was acidified with 1 N HCl and distributed
between Et2O and a saturated solution of salt. The Et2O phase
was evaporated, and the extract was obtained. GC analysis,
with 1 as an internal standard, was used for the quantitative
analysis of metabolites. Under aerobic conditions, 8, 8, and 7
mg of metabolite were obtained at 12, 24, and 48 h, respec-
tively; and under aerobic conditions, 9, 8, and 8 mg of
metabolite were obtained at 12, 24, and 48 h, respectively.